Analytical biochemistry system with robotically carried bioarray

ABSTRACT

An analytical biochemistry system featuring a substrate with reactants immobilized thereon at fixed, known locations, a holder supporting the substrate and a manipulator for transporting the holder to a fixed sample and to an inspection station. The reactants are binding agents for a target biomolecule in a sample which forms a bound substance having a detectable characteristic. The holder may be a standard pipettor, optionally carried by a robot arm or hand as the manipulator to contact the sample for detection of the presence of target biomolecules within the sample. In one embodiment, the holder is a pipette tip within which the substrate is housed, or it may be a pipette adapter which bears the substrate and fits within the sample wells of a standard microtiter plate.

This application is a continuation of U.S. patent application Ser. No.11/073,288 filed on Mar. 4, 2005 which is a continuation of U.S. patentapplication Ser. No. 10/200,720 filed on Jul. 22, 2002, which is adivisional application of U.S. patent application Ser. No. 08/586,116filed Jan. 16, 1996 now U.S. Pat. No. 6,660,233. All these applicationare incorporated by reference in their entirety for all purposes.

TECHNICAL FIELD

This invention relates to a system and methods for detecting thepresence of target biomolecules within samples with robotic assistancefor a sample holder carrying an array of reactants.

BACKGROUND ART

Assays for the detection of target biomolecules within a sample,especially of multiple target biomolecules within a sample, are oftenperformed by applying a volume of the sample to a test slide, membrane,or other substrate having immobilized reactants which may interact withthe target or targets to form detectable complexes. These immobilizedreactants are usually disposed at fixed locations, with samples broughtto these locations. U.S. Pat. No. 5,139,743, for example, discloses abiochemical analysis apparatus wherein an applicator takes up a liquidsample and applies the sample to a fixed position test film for chemicalanalysis of the sample.

Sometimes complexes of target biomolecules and reactants are visuallydetectable directly after an appropriate incubation period for thesample and reactants, or after numerous development steps whereindevelopment chemicals, such as fluorescent dye-conjugated molecules, areallowed to interact with the complexes. For example, the detectionmechanism in U.S. Pat. No. 5,296,194 involves optically detecting acolor change in a blood drop applied to a test slide.

U.S. Pat. No. 4,877,745 discloses methods for preparing immobilizedreagents and applying samples to immobilized reagents. In particular,this patent discloses dispensing precisely controlled volumes ofdroplets onto a medium at precisely controlled locations, to form arraysof immobilized reagents by a jet head. An x-y plotter may be modified tocarry a jet head so that reagent may be dispensed over an area.

Robotic laboratory workstations, such as the Biomek 1000 and 2000 ofBeckman Instruments, Inc. have been developed for automatically carryingout assays involving multiple reactants and multiple samples. Typicallysuch workstations are designed to deliver robotically precise volumes ofreactants to a number of different samples located at known areas withinthe workstation. Alternatively, workstations can robotically movesamples to reagents.

U.S. Pat. No. 5,171,537 to Wainwright et al. teaches activatedimmunodiagnostic pipette tips. The pipette tip houses a sphericalelement which is coated with a single ligand having affinity for atarget molecule of a sample. With this device, the test element may bebrought to contact the sample, as by aspirating the sample into thepipette tip. These pipette tips are limited in their sample throughputbecause they house only a single ligand reagent and thus preclude thedetection of multiple analytes within a sample.

A class of devices known as optical biosensors, characterized byimmobilized assay species within a supporter and a light collectiondevice coupled to an optical waveguide, is also known. Opticalbiosensors may be used for detecting and quantifying the presence ofspecific species in test fluid samples, such as in clinical diagnosticreactions. For example, U.S. Pat. No. 4,857,273 discloses an opticalbiosensor for immunoassays and certain other reactions. Other examples,involving use of an optical fiber, are U.S. Pat. No. 5,143,066 and U.S.Pat. No. 5,401,469.

It is an object of the present invention to provide apparatus andmethods for rapidly and automatically determining the presence ofmultiple target biomolecules in a single sample. It is another object ofthe present invention to provide analytical methods which requireminimal sample volume and a minimal number of liquid transfers. It is afurther object of the present invention to provide a device and systemfor rapid assessment of samples for target biomolecules which is readilyadaptable to a variety of chemical and other detection schemes.

DISCLOSURE OF THE INVENTION

The present invention achieves the above objects by providing ananalytical biochemistry system for automated analysis of samples for thepresence of target biomolecules. The system includes a solid substratewhich is supported by a holder and carried by a manipulator, such as arobotic arm. Immobilized on the solid substrate surface at discrete,site-specific locations are reactants in an array which are capable ofbinding with target biomolecules in specific binding reactions to formimmobilized biomolecule complexes. Such an array is termed a “bioarray”.The presence of target biomolecules in the sample is determined bydetecting immobilized biomolecule complexes on the bioarray with somekind of probe, e.g. a fluorescence detector. In operation, themanipulator moves the bioarray to contact the substrate surface with avolume of sample. Then the manipulator moves the contacted bioarray to adetection station to detect the absence or presence of immobilizedbiomolecule complexes. In alternative embodiments the bioarray isstationary and a sample manipulator moves samples to the holder. In thepreferred embodiment, the bioarray is mobile, being carried by amanipulator. A detection station is located near the sample to probe thesubstrate after interaction between the reactants and sample or sampleshas occurred.

Distinct reactants specific to different target biomolecules areimmobilized on a preferably flat, non-porous substrate. These reactantsform a plurality of active sites on the substrate at known locations.The substrate may be a planar strip with linearly-arranged reactantsforming separable spots or bands, or may be a planar sheet having anarea-wide arrangement of reactants, forming spots or dots in atwo-dimensional array, or may be a fiber or rod with substrate disposedin a manner similar to a strip.

The holder supports the bioarray and is carried by the manipulator whichtransports the substrate to the location of the fixed sample, and thento the location of the detection assembly. As stated, the substratecould be fixed and the sample transported. One example of a holder is apipette or a pipette tip, within which a bioarray is affixed. The sampleis drawn up into the pipette tip, as with aspiration from a bulb orvacuum pump, or withdrawal of a plunger. The sample is thus placed incontact with the substrate, allowing any target molecules which may bepresent within the sample to interact with the appropriate reactivesites on the substrate. After the appropriate incubation or reactionperiod, the sample may be removed from the pipette tip, as by airpressure or positive displacement with a plunger.

Another example of a useful holder is a pipette adapter resembling atruncated pipette tip and having a bracket or a flat surface orsupporting the substrate. The pipette adapter may be placed directlyinto a sample, such as in a well of a microtiter plate or in a vial, inorder to provide contact of the holder and the sample. The pipetteadapter and accompanying substrate are then removed from the sample to adetector station. The various holders of the present invention may beadaptations of standard pipetting tools. The holders also are designedto require minimal sample volumes and to allow optical inspection of thesubstrate with minimal interference by the holder.

The method for detecting target biomolecules within a sample includesthe steps of treating a substrate with a plurality of distinct reactantsto form reagents immobilized on the substrate at fixed, known positionsdefining an array, i.e. a bioarray. The reactants are selected to bindone or more target molecules to form a complex having a detectable andidentifiable character-istic such as a fluorescent signature. Thebioarray is supported in the holder. In turn, the holder has a shapewhich can be picked up by a manipulator which moves the substrate forcontact with the fixed sample, and then removes and possibly rinses thesubstrate at another location to remove unbound biomolecules. Then themanipulator moves the substrate to a probing station, such as an opticalinspection location for probing the active sites of the substrate with abeam for determining complementation of the target biomolecules bydetecting the optically detectable characteristic.

Inspection may include detection of fluorescence, light scattering,absorbance, reflectance, chemiluminescence, radioactive emission,conductivity or electronic property. Depending on the nature of thesubstrate, detection of transmitted light is also possible.

Prior to probing, intermediary steps to enhance visualization orrealization of complementation, such as treatment with developmentchemicals, fluorescent dyes, etc. may be desired. Optical inspection ofthe substrate within the pipette tip is possible by use of an opticalsurface on the pipette tip. Optical inspection on the pipette adapter isunencumbered. A manipulator in the form of a robotic arm gripping thepipette tip or pipette adapter type of substrate holder may place thebioarray in contact with the sample, and subsequently transfer thesubstrate to a detection assembly. Multiple sample transfers are thuseliminated. A computer controlling the robotic arm movement, theincubation times, and providing further analysis or display of detectedsignals from the substrate is preferred. An automated instrumentincludes a detection assembly, which in one embodiment includes a lasersource providing an excitation beam to impinge upon the active sites ofthe substrate, a light collector for gathering signals emitted from thesubstrate, and a detector, such as a photomultiplier tube or CCD array.Alternatively, it may have multiple detection assemblies, depending onthe requirements of the sample and the substrate chemistries. Relativemovement of an excitation beam and the Bioarray may be provided by therobotic arm holding the substrate or by scanning optics, such as a galvomirror, within the excitation path of the detection assembly. Asubstrate intended for use in the present invention may be anoligonucleotide array, a peptide array, or an immunochemical array,among others, and may be created on a separate member, such as a smallslide, and affixed to the holder, or it may be created directly on theholder. Creation of the bioarray may be via biopolymer synthesis on asolid phase member or deposition of reactants, e.g. by movable nozzles,such as the type used for ink jet printing, or by some other method. Thereactants may be affixed to the member via specific or non-specificcovalent linkages, physical adsorption, or some other form of adhesion.The interaction or complexing of the target biomolecules and theimmobilized reactants may be by affinity linkages, ionic linkages,adsorption, or some other reasonably secure manner. The presentinvention provides a simple, highly adaptable method and apparatus forquickly and easily assessing samples for the presence of biomolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an automated instrument for performingthe target biomolecule detection in accord with the system of thepresent invention.

FIG. 2 is a plan view of a linearly-arranged substrate for use in thesystem of FIG. 1.

FIG. 3 is a plan view of a two-dimensional substrate for use in thesystem of FIG. 1.

FIG. 4 is a plan view of a pipette tip having a substrate for use in thesystem of FIG. 1.

FIG. 5 is a plan view of a plunger-type pipette tip having a substratefor use in the system of FIG. 1.

FIG. 6 is a front view of a pipette adapter with bracket for use in thesystem of FIG. 1.

FIG. 7 is a side view of the pipette adapter of FIG. 6.

FIG. 8 is a perspective view of a pipette adapter, supporting asubstrate for use in the system of FIG. 1.

FIG. 9 is an end view of the pipette adapter of FIG. 8, showing detailsof the supported substrate.

FIG. 10 is a plan view of a pipette adapter positioned within the samplewell of a microtiter plate.

FIG. 11 is a front view of a flat bottom pipette adapter for use in thesystem of FIG. 1.

FIG. 12 is a perspective view of the flat bottom pipette adapter of FIG.11, showing a substrate at the base of the adapter.

FIG. 13 is an end view of a flat bottom pipette adapter of FIG. 11,showing details of the substrate.

FIG. 14 is a perspective view of the elements of an optical detectionstation for use in the system of FIG. 1.

FIG. 15 is a plan view of an alternate embodiment of the detectionstation for use in the system of FIG. 1.

FIG. 16 presents yet another alternate embodiment of the detectionstation for use in the system of FIG. 1.

FIG. 17 is a perspective view of a device which may be utilized forbiopolymer synthesis on a substrate to create a substrate in accord withthe present invention.

FIG. 18 presents a cross sectional view of the device of FIG. 17.

FIG. 19 is a plan view of a substrate and backing plate for biopolymersynthesis, showing a one-dimensional biopolymer array for a substrate inaccord with the present invention.

FIG. 20 is a plan view of a substrate and backing plate, showingtwo-dimensional biopolymer synthesis for a substrate in accord with thepresent invention.

FIG. 21 is a perspective view of a jet head-type reagent depositionapparatus for creating a substrate in accord with the present invention.

FIG. 22 is a plan view of the internal elements of a jet head of FIG.21.

FIG. 23 illustrates a method of affixing a substrate to a manipulator ofthe present invention.

FIG. 24 illustrates a first alternative method of affixing a substrateto a manipulator of the present invention.

FIG. 25 illustrates a second alternative method of affixing a substrateto a manipulator of the present invention.

FIG. 26 is a plan perspective view of a version of the instrument ofFIG. 1, modified to include a jet-head substrate treating apparatus.

BEST MODE FOR CARRYING OUT THE INVENTION

With reference to FIG. 1, a system 10 utilizing a movable bioarray isshown. Specifically, a robotic arm 12 carries a holder 20 which fits andtransports bioarray 11, first to the sample, which may be in well 17 ofmicro-titer plate 15 or in vial 16 of rack 14. Although a robotic arm isone form of manipulator which may be used, other simpler manipulatorsmay be employed, such as mechanical movements. A preferred type ofmanipulator device is the Biomek, a trademark for an instrument ofBeckman Instruments, Inc. As will be seen below, the substrate portionof bioarray 11 is mounted in a holder having a support region which maybe quite small. After the bioarray and the sample have had a sufficientincubation or reaction time for interaction of reactants on thesubstrate and any target biomolecules which may be present within thesample, the robotic arm 12 moves the substrate 11 to the detectionassembly 18 of instrument 10.

In FIG. 1, optical detection station 18 is presented in a cutaway viewshowing laser 19, light collector 21, and detector 22. Both thedetection station 18 and the robotic arm 12 may be attached to acomputer, not shown, which generates commands for movement of therobotic arm and receives signals from the detection assembly which may,in turn, be analyzed to determine whether a specific target biomoleculeis present and which may be displayed. The substrate itself is heldwithin a holder 20 which may be coupled to the cantilevered robotic arm12 via pick-up shaft 23 or by some other coupling method. Tracks in thetower 13, the arm 12, and base 59 of the instrument 10, as well ascontrols 24 within the robotic arm 12, position the pick-up unit 25relative to the samples and the detection assembly 18 with x, y, zmotion, i.e. three degrees of freedom. A wide range of motion isavailable over a base the size of a desktop. Many sample wells may bereached as well as many substrate holders having treated substrates oruntreated substrates which may be treated motion to a nearby locationwhere reactants may be sprayed or otherwise applied to the substrate.

FIG. 2 presents a linearly-arranged, flat substrate as a support portionof a bioarray, with active sites 30 forming bands along substrate 28which is shaped as a strip. Spaces 26 are provided to place theplurality of active sites 30 in a spaced-apart relation along sub-strate28. In FIG. 3, an area-wide treated substrate is created by positioningthe active sites in a two-dimensional relation on a planar sheetsubstrate. Active sites 30 are positioned in spaced-apart relation onsubstrate 28, as before. As illustrated in FIGS. 2 and 3, the activesites 30 may be bands, as in FIG. 2, or spots, as in FIG. 3, or someother shape. The spots are in known locations and are specific for atarget biomolecule. A plurality of linear substrates of FIG. 2 may alsobe arranged in parallel to create a two-dimensional bioarray. The sizeof the substrate is typically a few centimeters on a side, but could besmaller or larger.

By way of example, the reactants forming the active sites may comprisecomplementary DNA strands for detection by DNA hybridization or they maycomprise immunological biomolecules for detection by immunologicalcomplexing, such as formation of antigen-antibody com-plexes.

The device of FIG. 4 represents one example of a substrate holder whichmay be used to present the Bioarray to the sample. A pipette tip 27 isshown with a substrate 11 supported longitudinally along an inside wall.The substrate is preferably positioned along an inside wall of thepipette tip and the pipette tip is comprised of an optical glass orplastic allowing optical inspection of the substrate while the substrateis positioned inside the pipette tip. The sample is drawn into thepipette tip by aspiration and allowed to interact with substrate 11.Pipette tip 27 preferably has at least one flat surface, i.e. thesurface opposite the substrate, for accurate optical inspections. Thisfeature and a narrow bore also help minimize the amount of samplenecessary and place the sample and substrate in close proximity. Thepipette tip 27 may be used in conjunction with a rubber bulb, vacuumpump, robotic pipettor as in FIG. 1, or other device. The term “pipettetip” is meant to include pipettes, such as long cylindrical glass orplastic pipettes which are designed to operate in connection with asimple suction device.

FIG. 5 shows a second embodiment of a substrate holder wherein thesubstrate 11 is housed within a plunger-type pipette tip, in the samemanner as the FIG. 4 embodiment. Pipette tip 29 also has a narrow boreand flattened surface. The sample is drawn into the plunger-type pipettetip 29 through withdrawal of plunger 31. Positive displacement of thesample is used to eject the sample from the pipette, as by depression ofplunger 31 or by some other fluid manipulation. The embodiment of FIG. 5has an advantage over the embodiment of FIG. 4 in that the sample doesnot drain from the pipette tip when the pipette tip of FIG. 5 isdetached from a pipetting tool.

In FIGS. 6-9, a substrate holder takes the form of a pipette adapter 32characterized by a bracket 33 at one end. The bracket has opposed prongs36 a-b, easily visible in FIG. 8, which support the ends of thesubstrate which is part of the bioarray. The opposite end of thebracketed pipette adapter preferably has a coupler 37 for joining arobotic or standard pipetting tool.

In FIG. 8, the coupler 37 is depicted as a hollow cone which may fit theconical shaft of a robotic or standard pipetting tool with anappropriate securing mechanism, such as a friction fit, with provisionfor ejection of the adapter for use. Many different types of couplersmay be used, however. Of course, the pipette adapter 32 need not haveany coupler, but it is preferred that the adapter have a gripper orother means for manipulating the adapter so, for example, the adaptermay be moved into and out of sample wells easily.

The bioarray is preferably oriented so that the active sites facedownward. Thus, when the adapter is placed within a sample well, as inFIG. 10, contact of the active sites of the substrate and the sample isfacilitated. Pipette adapter 32 is preferably equipped with knobs 35 onthe prongs of the bracket, visible in FIGS. 6-9. These knobs positionthe substrate slightly above the bottom of the sample well, and protectthe treated substrate from physical abrasion and contamination. Withoutsuch knobs, placement of the adapter into the well may press thesubstrate so close to the bottom of the well as to exclude sample fromthe face of the substrate, preventing proper contact and interaction ofthe sample and the substrate. Bracketed pipette adapter 32 alsopreferably contains a ring or disk-shaped evaporation barrier 34 whichis disposed about the midsection of the adapter, or at the base of thebracket portion. Because some samples may easily evaporate, evaporationbarrier 34 is preferably provided to protect the sample during thesubstrate and sample incubation period. In FIG. 10, evaporation ring 34is seen providing a barrier when pipette adapter 32 is inserted intowell 17, thus limiting the exposure of sample 38. The diameter ofpipette adapter 32, and particularly bracket 33, is sufficiently narrowin order to easily fit within the microtiter plate's well, as also seenin FIG. 10.

FIG. 9 presents an end view of the bracketed pipette adapter 32. Thisview is indicated by axis 9-9 of FIG. 6, viewed in the direction of thearrows. From the end view, the substrate 11 is more clearly visible inits preferred downward facing orientation. The substrate of FIG. 9 is ina linearly arranged, but segmented, form. Thus strips 11 a, 11 b, and 11c are positioned in a generally parallel arrangement and secured byprongs 36 a-b of the bracket 33. The nature and shape of the substratemay be easily adapted to sample, applicator, and space considerations.The two-dimensional substrate of FIG. 3 would adapt easily to bracketedpipette adapter 32. The substrate is held by prongs 36 a-b by adhesion,welding, clamping, or any other means for gripping which will notinterfere with the testing of the sample. In the end view of FIG. 9,evaporation disk 34 is visible beyond the prongs and the substrate.

In FIGS. 11-13, a flat-bottom pipette adapter 39 is utilized to supportand transport the bioarray. Flat-bottom pipette adapter 39 has a flatbottom surface 40, visible in FIG. 12, and may have a coupler 37 at anend opposite to the flat bottom surface 40 for fitting the adapter topipetting tools or a robotic arm, as in the bracketed pipette adapterembodiment. Similarly, flat bottom pipette adapter 39 may have a simplemeans for manually gripping the pipette adapter and applying thesubstrate 11 to the sample. Also, as with the bracketed pipette adapter32, the flat bottom adapter 39 is preferably outfitted with knobs 35 andevaporation ring 34. In FIG. 13, the substrate 11 is seen to be atwo-dimensional array of spots or dots, as in FIG. 3. Evaporation ring34 is visible in this view, taken along axis 13-13 of FIG. 11, andsituated beyond flat bottom surface 40.

In FIG. 14, details of the internal elements of a biomolecule probestation are shown. Although various probe methods are available, theoptical detection station 18 is an example. The station may be part ofan analysis machine having a robotic arm, such as that shown in FIG. 1.The robotic arm, pipetting tool, or other substrate holder positions thesubstrate, after it has interacted with the sample, in the path of alaser beam. Laser 41 creates a beam which impinges upon the active sitesof the substrate 11, which is held within bracketed pipette adapter 32.The wavelength of the beam is selected to cause the return of aradiation signature from target molecules bound to the substrate. Such asignature comes from an optically detectable characteristic radiationpattern of the bound target molecules when excited by radiation of thebeam, such as a characteristic band of fluorescent wavelengths. Timegated fluorescence, or other optical signal enhancement techniques, mayoptionally be used. The incident beam from the laser is scanned acrossthe active sites of the treated substrate by relative motion of thesubstrate and the beam. Light emitted from the active sites is collectedby light collector 42 and directed to detector 43, which may be aphotomultiplier tube, CCD array, or other detection device, and which ispreferably associated with a computer for any further analysis ordisplay of the signals received from the substrate. Additional opticalelements, such as wavelength selective filters, may be disposed ineither the incident beam or the return light, as required by thecharacteristic radiation signature. Scanning may be accomplished bymoving the substrate relative to the laser beam, by utilizing a scanningreflector such as a galvo mirror or polygonal mirror, or by some otherwell known means. Alternatively, the area of the laser beam may beexpanded such that the entire area of the array is illuminatedsimultaneously, and scanning is not required.

The bioarray is optically probed by the beam for determining the extentof complexing of the reactants in the active sites of the substrate withtarget biomolecules in the sample. The optical inspection may be forfluorescent signals, reflectance, absorbance, light scattering, orchemiluminescence, among others. Details of the optical system may varyaccording to the nature of the signal to be detected. FIG. 14illustrates a substrate 11 within bracketed pipette adapter 32 andfacing in a downward orientation for impingement by the laser beam. Thisarrangement of the elements of the detection assembly is presented as anexample of the arrangement of the detection assembly 18 of FIG. 1. Ineither case, the robotic arm may easily move the substrate and theassociated bracketed pipette adapter to the detection assembly after theappropriate sample incubation period. The robotic arm may, however, becapable of moving the substrate so that it is oriented vertically, or insome other manner, relative to the laser beam. Also, the laser sourcefor the excitation path may be positioned in a manner other than shownin order to impinge upon the substrate. Optical fibers may be employedto direct the beam or the return signal.

FIG. 15 presents a detection arrangement for probing of a substratewithin a pipette tip. Pipette tip 27 has an optical surface so as not tointerfere with the optical inspection. As with the bracketed pipetteadapter, laser 41 impinges upon the substrate 11 and emitted signalsfrom the substrate are gathered by light collector 42 and directed todetector 43 where the signals may be sent onto a computer for furtheranalysis. The excitation beam from the laser impinges on the substratethrough the wall of the pipette tip. Arrow A of FIG. 15 indicates oneexample of how the substrate may be scanned, i.e. by providing avertical motion to the pipette tip, via the robotic arm or some othermechanism. In FIG. 16, the substrate 11 within pipette tip 27 is scannedby the incident laser beam through the action of a scanning reflector44, which may be rotated in direction B to cause scanning of thesubstrate 11. An automated apparatus such as instrument 10 of FIG. 1 mayhave a plurality of detection assemblies to which the robotic arm maymove the substrate for reading, depending upon the type of manipulatorused for the substrate, the type of signals to be read from thesubstrate, and the nature of the substrate and target biomoleculesthereon. The substrate may be formed by the device shown in FIGS. 17-20,which allows biopolymer synthesis on a solid support. The substrate maybe used directly, or the biopolymers created by the device may becleaved from the substrate and affixed to another substrate in anydesired format. The method and apparatus depicted in FIGS. 17-20 is thesubject of commonly-assigned U.S. Pat. No. 5,429,807, which isincorporated herein by reference. The device of FIG. 17 presents asynthesis device 45 which is a thick block having a plate surface 46within which are a plurality of grooves or channels 50. Channels 50 areconnected to tubing 47 at the underside of block 45 for flowing reagentsthrough channels 50. The cross sectional view of FIG. 18, taken alongaxis 18-18 of FIG. 17, more clearly illustrates the reagent flow throughthe block herein tubing 47 a is connected to an inlet tubing connector49 which communicates with channel 50. Tubing 47 b communicates withoutlet tubing connector 52 which in turn communicates with channel 50.Thus, reagents may be caused to flow through any of the channels 50.

To perform a synthesis, a solid support material, such as a sheet ofactivated polypropylene, may be placed on top of the channels of theblock. A backing plate may be used to sandwich the polypropylenesubstrate, allowing the flexible polypropylene to seal against thechannels 50 of the block 45. The backing plate 52 of FIGS. 19 and 20 mayhave holes 53 which may be aligned with holes 48 of block 45. Thebacking plate and the block may then be secured to one another.Synthesis or biopolymerization may be performed by activating thesurface of the substrate, if necessary, and by flowing reagents throughthe channels, to cause formation of strands of biopolymers anchored tothe substrate. This results in a one-dimensional array of biopolymers54, as seen in FIG. 19. If desired, the block may be repositioned withrespect to the substrate 55 and the process repeated. In FIG. 20.one-dimensional arrays 54 a-b are presented in 90° offset orientations.The areas of overlap 56 provide new biopolymers having elements of eachone-dimensional array 54 a-b. Indexing pins 58, visible in FIGS. 19 and20, may be utilized to position the substrate 55 in relation to theapplicator. Indexing pins 58 mate with holes 57 in block 45. Theresulting arrays may be utilized as is, or may be cleaved from thepolypropylene substrate 55 and affixed to some other support.Additionally, the substrate 55 having the arrays may be segmented andattached to substrate 28, in the manner of FIGS. 8 and 9. Althoughchannels 50 are illustrated, cavities for reagent flow having some othershape may be used. Although polypropylene is presented in the abovediscussion, other substrates such as glass, Pyrex, silicon, polystyrene,etc. may be utilized as supports for synthesis, as suggested in PCTapplication No. WO93/09668.

With reference to FIG. 21, another method of creating a substrate isshown, utilizing movable nozzles. A support 28 is positioned on supportframe 65 within a spray station. A plurality of ink jet-type heads 60with nozzles 61 at the spray station are used to selectively depositreactants on support 28 to create the plurality of active sites. Suchheads are well-known in the field of ink jet printing. In the presentinvention, such heads are adapted for dispensing the reactants onto thedesired locations of the substrate. If necessary, the substrate may beactivated to receive and immobilize the reactants.

In FIG. 21, a plurality of different jet heads 60, each having a nozzle61, may be moved on respective rails 62 in the direction indicated byarrow C. Each of the jet heads 60 dispenses a different reactant. Motioncontrol of the jet heads 60 along rails 62 may be provided by acomputer. The array may be moved by an actuator 63, which causes an arm64 to move support frame 65 carrying the support 28. The actuator 63 maybe a linear motor similar to that used to move magnetic heads in discdrives. In the situation where the substrate for the treated substrateis a strip, fixed position jet heads may be desired.

FIG. 22 illustrates a typical ink jet-type dispensing head as applied inthis invention. Reactant contained in reservoir 67 passes through supplytube 69 to piezoelectric pumping chamber 66 and through chamber 66 tonozzle 69. Electrical pulses applied to pumping chamber 66 cause it toexpand and contract in volume. Each time a pulse is applied and removed;this expansion and contraction event ejects a droplet 70 of reactantfrom the nozzle. Additional details of the design and operation of sucha reactant dispensing device are disclosed in previously referenced U.S.Pat. No. 4,877,745. In addition to dispensing reactants, such adispensing head might be employed to dispense ink or dye onto thesubstrate to form barcode patterns for machine reading of theidentification of a bioarray.

Another method of preparing the bioarray is by a technique analogous toa printing method. In this technique an analyte is deposited on asubstrate by stamping or embossing a very thin layer with an array ofanalyte spots at desired locations. For example, an antigen attached toa molecule anchored to the substrate by pressure contact will combinewith an appropriate antibody associated with a specific targetbiomolecule. The antibody may be fluorescent for optical detection.Other methods of preparing the substrate may be used, particularlyphotolithographic techniques. In a journal article entitled“Light-Directed, spatially Addressable Parallel Chemical Synthesis” byS. Fodor et al. in Science, Feb. 1-5, 1991, p. 767, the authors describea method of synthesizing complex compounds at spatially discretelocations defined by photomasks of the type used in the semiconductorindustry. Molecular building blocks are deposited at desired locationsby exposing underlying building blocks, i.e. “deprotecting” heunderlying block for a reaction with the superposed building block.Successive building blocks are added until a desired compound is formed.The location of each compound is precisely known from the mask set andthe sites may be very closely spaced, limited only by the diffraction oflight.

A method of imaging, i.e. probing, a substrate having microscopicfeatures is by means of condensation figures (CFs) described in ajournal article entitled “Imaging of Features on Surfaces byCondensation Figures” by C. Lopez et al. in Science, Apr. 30, 1993, p.647. The authors describe the formation of an array of tiny droplets ona cold surface having an array of spots which are not wet by thedroplets. The spots could be the complex compounds described in thepreceding paragraph. The droplets are observed with microscope optics.

Still another bioarray forming technique is described in an article byB. Healey et al. in Science, Aug. 25, 1995 p. 1078. The authorsdeposited microscopic polymer arrays on a flat substrate by depositing alayer of polymerization solution on a flat plate, such as a glass chipwhich had been activated for adhesion with the solution. A bundle offibers was brought into contact with the solution and then backed offand the substrate rinsed. Light was directed into the non-contacted endof the fiber bundle to cause polymer deposition on the substrate belowthe fibers of the fiber bundle. Polymer spots of a 2.0 micrometerdiameter and a spacing of 4.0 micrometers were produced. Yet anotherbioarray forming technique is the Southern blotting method in whichhybridization is used simultaneously on a large number of DNA segments.DNA is fragmented, electrophoresed, denatured and transferred from a gelto filter paper. Positions of numerous fragments are established. TheDNA fragments are robotically moved in accord with the present inventionand combined with radioactive phosphorous labeled RNA which can beidentified. The degree of DNA-RNA complementation, i.e. probing of thesample, can be determined by autoradiography.

In another bioarray forming technique a polyunsaturated polymerizedlipid layer is applied to a support. The lipids have a member of aspecific binding pair bound to one end. The lipids have an opticalcharacteristic which is modified upon complexing the other member of thebinding pair. Such an optical characteristic can be polarization oflight and such light is used to probe the bioarray.

In FIG. 23, affixing of bioarray 11 to the flat bottom surface 40 offlat bottom pipette adapter 39, seen in FIGS. 10-12, is given as anexample. Specifically, in FIG. 23, a two-dimensional bioarray 11 ispreformed and then attached to the flat bottom surface 40 of pipetteadapter 39. FIG. 24 presents an alternative wherein the substrate 28 isaffixed to flat bottom surface 40 of the pipette adapter, beforereactants are caused to become immobilized on the substrate to form thebioarray 11. This alternative works particularly well with the printingtype of creation for substrates as discussed with reference to FIGS. 21and 22. In FIG. 25, it can be seen that the bare substrate 28 may be anintegral part of the adapter as fabricated, for instance by injectionmolding. The substrate 28 is then activated, indicated by shading inFIG. 25, and then the reactants are deposited or otherwise caused toattach to substrate 28.

Although the method of the present invention is designed for detectionof target biomolecules in a sample, quantification of the targetbiomolecules is possible by, for example, recording the sample volumeexposed to the substrate, quantifying the degree of complementation atthe active sites of the substrate, and calculating the amount of targetbiomolecule present from these two values. Quantification of the degreeof complementation may be performed, e.g., by measuring the percentageof active sites which are fluorescently-labeled or give some otheroptical signal indicating complementation. Additionally, affixing anexcess amount of reactants to the substrate compared to the amount ofsuspected target biomolecules of the sample is a preferred practice andmakes quantification more accurate.

Referring to FIG. 26, an alternate embodiment of instrument 10 of FIG.1, incorporating the jethead-type reagent dispensing means, is shown. Asbefore, the instrument contains a detection assembly 18 and has alocation for placement of the samples. In this case, the samples arewithin wells 17 of microtiter plate 15. Additionally, FIG. 26 shows alocation for a holder 20 for a substrate. For simplicity, pipetteadapter 32 has a bioarray held within opposed prongs of a bracket, asillustrated in FIGS. 6-9. Specifically, pipette adapter rack 81 is shownhaving a plurality of pipette adapters 32 a. Here, the instrument alsocontains a jethead dispensing device 80. The dispensing device is of thetype discussed with regard to FIGS. 21 and 22. As with the instrument ofFIG. 1, this variation has a robotic arm 12 attached to a tower 13which, in turn, is attached to a base 59. As before, tower 13 and base59 have tracks for providing both vertical and horizontal motion to therobotic arm. Additionally, in FIG. 26, the detection assembly 18 anddispensing device 80 are depicted as blocks having holes. These blocksillustrate that the instrument is provided with various stations, eachhaving dedicated operations. The holes enable access to the holder whichis attached to the robotic arm.

In operation, the instrument of FIG. 26 provides motion to the bioarrayvia the robotic arm which picks up a pipette adapter having a substratesupport from rack 81, and then moves the pipette adapter into dispensingdevice 80. Within dispensing device 80, the support may be activated, ifnecessary, and dispensing or printing of reactants upon the support tocreate a substrate occurs. Then, robotic arm 12 moves the pipetteadapter from dispensing device 80 into contact with a sample, as byplacing pipette adapter 32 within a sample well 17 of microtiter plate15. The pipette adapters 32 b shown in the microtiter plate 15 have beendeposited into the wells by robotic arm 12 for interaction of thesubstrates and the samples. After the appropriate incubation time,robotic arm 12 picks up each pipette adapter and moves it to detectionassembly 18 for detection, as before.

In the above description, the robotic arm moved the pipette adapter,with holder and bioarray, to a sample location, such as a microtiterplate. However, the robotic arm could pick up sample in a pipettor andbring it to a stationary holder where the pipettor could dispense thesample onto the holder. Then, the same robotic arm, or another one, withan appropriate gripper could move the holder to a detection station.

The detection station could be any of the optical types described above,but could also be a radioactive tag detector if the immobilizedreactants for the target biomolecule had been radioactive. Also, if thetag was a moiety suitable for detection by laser desorption massspectrometry (LD-MS), then an LD-MS detection system could be used.Other tags and detection systems will be evident to those skilled in theart.

1. A system for detecting the presence of a target biomolecule within asample comprising: a support surface treated with distinct reactantsimmobilized thereon in a spaced-apart relation, forming a substratehaving a plurality of spaced-apart active sites, at least one of thereactants being reactive with a target biomolecule to form a boundcomplex having a detectable characteristic, holder means for supportingthe substrate, an inspection station having means for probing thespaced-apart active sites of the substrate for said detectablecharacteristic of the sample, and manipulator means for bringing theholder means into contact with the sample and into said inspectionstation. 2-76. (canceled)
 77. A method of reading an array of moietieson at least a portion of a surface of a transparent slide which isopposite a first portion on an opposite surface, which array has beenpreviously exposed to a sample, the method comprising: (a) exposing thearray to a sample by placing the slide in a vapor tight barrier; (b)transferring the slide to an open environment for washing and drying ofthe array; (c) mounting the slide on a slide holder and retaining theslide thereon in an enclosed and protected environment without the arraycontacting the holder; and (d) inserting the holder into an array readerand reading the array.
 78. A method of reading an array of moieties onat least a portion of a rear surface of a transparent slide which isopposite a first portion on the front surface, which array has beenpreviously exposed to a sample, the method comprising: (a) exposing thearray to a sample by placing the slide in a vapor tight barrier; (b)transferring the slide to an open environment for washing and drying ofthe array; (c) mounting the slide on a slide holder and retaining theslide thereon in an enclosed and protected environment in which thepreviously exposed array faces, and is spaced apart from, a backermember of the holder without the array contacting the holder; and (b)inserting the holder into an array reader and reading the array.
 79. Amethod according to claim 78, wherein the moieties are polynucleotidesof different sequences.
 80. A method according to claim 79, wherein themoieties are DNA of different sequences.
 81. A method according to claim78, wherein the array is read through the front side of the slide.
 82. Amethod according to claim 81, wherein the array reading comprisesdirecting a light beam through the slide from the front side and ontothe array, and detecting a resulting signal from the array which haspassed through the slide and out the slide front side.
 83. A methodaccording to claim 77, wherein the holder has front and rear clamp setswhich can be moved apart to receive the slide therebetween, and whereinthe slide is retained in the enclosed and protected environment by theclamp sets being urged against portions of the front and rear surfaces,respectively.
 84. A method according to claim 78, wherein the holder hasfront and rear clamp sets which can be moved apart to receive the slidetherebetween, and wherein the slide is retained in the enclosed andprotected environment by the clamp sets being urged against portions ofthe front and rear surfaces, respectively.
 85. A method according toclaim 77, wherein the holder has: a body having side portions and achannel intermediate the side portions and extending in a directionbetween ends of the body; and front and rear clamp member sets withmembers disposed about the channel, one set of which is fixed to thebody side portions while the other set is movable to an open positionaway from the fixed set; and, wherein the slide is retained in theenclosed and protected environment by being urged against the fixedclamp member set.
 86. A method according to claim 78, wherein the holderhas: a body having side portions and a channel intermediate the sideportions and extending in a direction between ends of the body, thebacker member, comprising a bottom surface of the channel; and front andrear clamp member sets with members disposed about the channel, one setof which is fixed to the body side portions while the other set ismovable to an open position away from the fixed set; and wherein theslide is retained in the enclosed and protected environment by beingurged against the fixed clamp member set.
 87. A method according toclaim 86, wherein the clamp sets are resiliently urged toward oneanother, and wherein the movable set is moved to the open position priorto mounting the slide on the holder.
 88. A method according to claim 87,additionally comprising a control member set positioned on the holderoutside the channel and wherein the control member set is moved to movethe movable clamp set to the open position.
 89. A method according toclaim 88, wherein the front clamp member set is fixed to the body sideportions and the rear clamp member set is movable.
 90. A methodaccording to claim 89, wherein the control member set is moved rearwardto move the clamp member set to the open position.
 91. A methodaccording to claim 78, wherein the holder additionally has a body havinga channel with a closed end, wherein the backer member comprises abottom surface of the channel; and wherein the mounting of the slide onthe holder comprises sliding the slide in an endways direction of thechannel and into the enclosed and protected environment in which aleading end of the slide abuts the closed end of the channel.
 92. Amethod according to claim 87, wherein members of each of the front andrear clamp member sets are disposed on opposite sides of the channel,and wherein the mounting of the slide on the holder comprises, when theclamp member sets are in the open position, sliding the slide in anendways direction of the channel between the clamp member sets and intothe enclosed and protected environment.
 93. A method according to claim92, wherein the holder additionally has two spaced apart guidesextending from the body adjacent respective sides of the channel, andwherein the slide is slid into the enclosed and protected environmentalong the guides and in which enclosed and protected environment atrailing end of the slide is positioned between the guides.
 94. A methodaccording to claim 93, wherein during the mounting of the slide portionsof the slide front and rear surfaces are gripped and the grippedportions used to then slide the slide into the enclosed and protectedenvironment, which gripped portions are positioned between the guideswhen the slide is in the enclosed and protected environment.
 95. Amethod according to claim 94, additionally comprising removing the slidefrom the enclosed and protected environment, the removing comprisinggripping portions of the slide front and rear surfaces which are betweenthe guides and using the gripped portions to slide the slide in anendways direction opposite to that during the slide mounting.